Protein recognition of specific DNA-binding sites is critical for the proper biological function of all nucleated cells. Binding specificity is a complex process ultimately dependent upon a protein's ability to complement either directly or indirectly, a DNA sequence. Often, small molecule effectors, which act as environmental sensors, must first bind to the protein to induce conformational changes necessary for specific DNA- binding. Recognizing such environmental signals is crucial for precise transcription regulation and thus requires effector binding to display high specificity. In oligomeric proteins, effector binding is often cooperative. To understand, at the structural level, the mechanism by which environmental signals are interpreted at the level of transcription, we have undertaken crystallographic studies on the E. coli Purine Repressor protein, PurR. Interestingly, the 289 amino acid residue binding domain of the PurR purine effector appears to have its evolutionary origin in the monomeric Periplasmic Binding Domains. Moreover, PurR belongs to a large, structurally conserved, family of oligomeric bacterial gene regulators, the LacI family. The PurR structure will be invaluable to fully understanding the biology of the LacI family, of which none have had their three dimensional structures determined. Therefore, this proposal has several long-term objectives that are designed: to elucidate the structural mechanism by which PurR recognizes a high affinity DNA-binding site; to broaden our understanding of DNA recognition by comparing the PurR-DNA complex to other protein-DNA complexes; to assess the effects of purine-binding on the conformation of PurR; to provide a structural basis to the cooperative nature of purine binding to PurR; and to delineate the structure-function relationships between PurR and the I-ad family as well as the Periplasmic Binding Proteins. To fulfill these long-term objectives, this proposal has four specific aims: (1) to determine the high-resolution crystal structure of the ligand-free Corepressor Binding Domain, CBD, of PurR; (2) to determine the crystal structures of CBD bound to hypoxanthine and guanine; (3) to crystallize and determine the structures of "mutant" CBD proteins which show diminished corepressor binding; (4) to determine the high-resolution crystal structure of a PurR- Hypoxanthine-purF complex.